1. Field of the Invention
The invention lies in the art of biomedical, scientific instrumentation. More specifically the invention relates to cytometers and instruments for high speed identification and sorting of cells, organelles and chromosomes. In particular the invention discloses an improved cytometer event charging and sorting apparatus and method.
2. Description of the Prior Art
Various techniques of flow cytometry have been employed over the last two decades from an initial effort to count particulate matter in a fluid environment, subsequently to size particles and more recently to quickly quantify multiple chemical, physical or structural properties of cells and cellular composites of inhomogeneous populations. The first such effort related to counting individual red cells in a liquid suspension forced through a capillary glass tube on a microscope stage. Problems encountered by such means involved standardizing capillary tubes, assuring proper focus, maintaining even flow and obtaining appropriately sensitive photoelectric apparatus to accomplish an accurate count.
Some of these problems were resolved by injecting the particle suspension into a laminar sheath flow of fluid, which flow surrounded and aligned the particles, and thereby virtually eliminated large particle blockage and coated the particle stream. Particle count by such means was accomplished by detecting the variation of electrical characteristic of the path through the laminar flow caused by the inclusion or exclusion of cellular matter therein. In addition particle sizing could be accomplished because pulse amplitude width was related to particle volume, and could be evaluated by pulse-height analyzers or nuclear pulse amplifiers. Photoelectric counting was later introduced. Subsequent cytometric application utilized spectrophotometry to quantify cellular constituents or alternatively to clarify cellular constituents via multiple simultaneous measurements of different cellular features, through UV absorption and photon scattering.
All the foregoing systems required a suspension of cells to pass through a constricted channel traversed by a beam of light orthogonal to said channel in which light intensity varied dependent upon position of the cell in the channel. Another possible variation, however, directed the light beam parallel to the flow and made calculations based on light scatter. Florescence at variable wavelengths or absorption characteristics were later used to characterize DNA and RNA constituents in the flow orthogonal to the illuminating beam.
Later cytometric improvements involved pneumatic, hydraulic and electrostatic techniques to separate cells from a flow after photometric or electrical sensing. Another variation utilized a fluid switch cell sorter which diverted a stream by means of a sonic transducer that converted laminar flow to turbulent flow.
More recent efforts, however, utilize a sheath fluid flow chamber to which is centrally added a fluid flow of sample body cells or organelles in aqueous suspension. Sample cells or constituents thereof in aqueous suspension are labeled with fluorescent dye molecules that bind specifically to the constituents to be measured.. For example DNA may be stained with propidium iodide or mithramycin, while other constituents may be labeled with (monoclonal) antibodies conjugated with some fluorescent dye such as FITC or phycoerythrin.
The flow chamber is vibrated at high frequency by a piezoelectric transducer which causes a sheath microscopic jet stream exiting the flow chamber with samples to break into discrete droplets from an exit point of the flow chamber. Carried by the microscopic jet of water, the cells on exiting the flow chamber pass one by one through an intense beam of excitation (laser) light in a measuring region of the flow cytometer.
Each cell or event thereby produces a short flash of fluorescence, the intensity of which is proportional to the cellular content of the fluorescently labeled constituent. The flashes of fluorescence are collected by appropriate optics which focus the light on a sensitive detector. The detector transforms the flashes of light into electrical pulses, which are measured and recorded by electronics and a computer. Each cell also causes scattering of the excitation light. Scattering is a function of size, shape, and structure of an event (cell). Thus, cellular content of several constituents, labeled with various dyes fluorescing at different wavelengths as well as size, shape, and structure can be recorded for each cell/event in large numbers. Upon exiting the flow chamber and before breaking into discrete droplets the jet is passed through electrical charging means that charge each droplet of interest either positively or negatively as determined by the above described laser identification of samples or events in the sheath flow prior to droplet formation. The charged droplets then pass through a pair of vertical plates, one charged at a negative voltage and the other at a positive voltage. The positively charged droplets shift stream toward the negative plate and the negative droplets shift stream toward the positive plate. Uncharged droplets continue in a straight line out of the flow chamber to a collector tube below.
Although the foregoing electronic charging of droplets allows for sorting of particles with two attributes per run by positive or negative charging, there remains a long standing need for faster identification of more physical or chemical characteristics than presently exist in the art, and therefore more accurate and more numerous sorting of events per run than is permissible with state of the art flow cytometers. Faster and more efficient cytometer methods are desperately needed because of recently encountered need for vast immunology studies. Flow cytometer technology in general makes it possible to distinguish subpopulations of cells as, for example, in analysis of asynchronously growing cell cultures, where cells in the different phases of the cell cycle are readily distinguished, or in immunology, where the flow cytometer discriminates between different subsets of lymphocytes.
The invention disclosed herein overcomes limitations of the prior art by substantially speeding up the cell event sorting process. As described above, prior art cytometers are limited to but two events (characteristics) per sort by charging events as each event leaves the flow chamber with either a positive or a negative charge. The invention herein describes a method and apparatus for applying multiple and variable charges to events as each event leaves the flow chamber and is identified and in addition permits a greater degree of focus and control of the plurality of diverted events/streams, thereby permitting a plurality of event categories or sorts per individual run, substantially in excess of existing art, and substantially increasing accuracy and efficiency.